Hololena curta venom a potent inhibitor of voltage sensitive Ca2+ channels and neurotransmitter release in mammalian brain, and synthetic funnel web spider toxin an inhibitor of P channels, were examined for their activity on autonomic nerves. Hololena curta (0.5 to 5.0 micrograms venom protein/ml) potently inhibited motor responses of the cholinergic guinea pig ileum myenteric plexus and the adrenergic rat anococcygeus muscle. Synthetic funnel web spider toxin was inactive at concentrations up to 100 microM. Hololena curta inhibited K+, and electrically evoked release of tritium from labeled superfused tissues. Furthermore, K(+)-contracted rat aorta was not relaxed by Hololena curta thereby precluding effects of Hololena curta on postjunctional L type smooth muscle Ca2+ channels. The pattern of effects of Hololena curta on peripheral autonomic nerves was similar to the N channel inhibitor omega-conotoxin GVIA. These results suggest that Hololena curta venom constituents block Ca2+ channels in peripheral autonomic nerves. The study failed to establish the presence of functional P type Ca2+ channels on these peripheral autonomic nerves and further suggests that N type channels may be exclusively responsible for supplying the Ca2+ necessary for neurotransmitter release in these nerves.